1. Field of the Invention
The present invention relates to a laser magnetic immunoassay method utilizing an antigen-antibody reaction and apparatus therefor, which enables quantitative measurement of a specified antigen or antibody in a very small amount of a sample. It relates also to a superparamagnetic material-labelled substance for use in such method and apparatus and to a method for the manufacture of same.
2. Prior Art
Development of immunoassay methods utilizing an antigen-antibody reaction is now being made on a global scale as an early detection method for new virus-based diseases such as AIDS and adult T-cell leukemia, as well as various cancers.
Examples of micro-immunoassay methods utilizing hitherto known primary reaction that have been put in practice include radioimmunoassay (RIA), enzyme-immunoassay (EIA), fluorescence-immunoassay (FIA), etc. These methods use antigens or antibodies which are labeled with an isotope, an enzyme or a fluorescent substance in order to detect the presence or absence of corresponding antibodies or antigens, respectively, that react specifically therewith.
Although the RIA has high sensitivity for detection, it has many restrictions in its practice because it uses radioactive substances as a label substance. EIA and FIA, both of which have less restrictions and thus are easier to practice than RIA, have low sensitivity for detection and are difficult to give accurate quantitative measurements.
In order to overcome the defects of the above-described immunoassay methods, the present inventors previously studied laser immunoassay methods which were based on different principle than the above-described methods and proposed laser magnetic immunoassay methods and apparatuses, and also methods for the preparation of specimens as described in WO/88/02118 (PCT/JP87/00694 corresponding to Japanese Patent Application Nos. 61-224567, 61-252427, 61-254164, 62-22062, 62-22063, 62-152791, 62-152792, and 62-184902), and Japanese Patent Application Nos. 62-264319, 62-267481, 63-6050, 63-102912, 63-102913, 63-102915, 63-102917, 63-102918, 63-156519, and 63-156520. In the laser magnetic immunoassay methods according to the prior proposals, magnetically labeled specimens are concentrated locally at a desired position or point of concentration in a gradient magnetic field using magnetic micro-particles and the laser beam is irradiated to the local concentration point to generate an output beam or light from the specimen such as interfered light, scattered light, transmitted light, diffracted light, etc., which is then detected by appropriate detecting means. The methods enable ultra-micro detection in the order of picograms without using isotopes.
In the laser magnetic immunoassay method as described above, a magnetic-labeled body or substance, which is composed of a micro-particle as a label and an antigen or antibody attached thereto, and an antibody or antigen, respectively, as a specimen or sample are subjected to antigen-antibody reaction to form a magnetic-labeled immunocomplex (or a magnetic-labeled body-specimen complex, as used in the previous applications), which is then separated and removed from unreacted magnetic-labeled body using magnetic force or centrifuging, followed by detection of the magnetic-labeled immunocomplex. The operation of separation and removal is relatively cumbersome and it will be very advantageous if it is possible to recognize only magnetic-labeled immunocomplexes.
Generally, labeling methods practically used nowadays including the above-described laser magnetic immunoassay method as well as RIA, EIA, FIA, etc., accompany troublesome washing step which must be repeated many times in spite of improved detection sensitivity. On the other hand, a micro-particle aggregation method (PA), which is one of the non-labeling methods, is disadvantageous in that it has a low sensitivity for detection in spite of the fact that the adjustment or preparation of specimens is simple and easy.
It is therefore very advantageous if labeling methods can be performed without separating and removing unreacted labeling materials and only specimens can be detected selectively.
Other examples of conventional non-separating type immunoassay methods utilizing magnetic micro-particles include the following ones.
(1) Toshimitsu Musha, Japanese Patent Application No. 60-257545, "Method for Detecting Immune Reaction Using Magnetic Field" describes a method in which polarized radiation is radiated onto magnetic micro-particles while they are being dislocated due to a change in the magnetic field and the occurrence of an immune reaction is detected by detecting the change in the plane of the polarization of scattered light generated upon antigen-antibody reaction of the micro-particles. The publication describes the measurement method itself in detail but fails to describe examples as to how to apply it to an antigen-antibody reaction practically and sensitivity for detection or a like is unknown.
(2) Yukio Karube and Chiki Ishii, Japanese Patent Application No. 61-130506, "Method for Measuring the Concentration of Antigens and Antibodies", and Japanese Patent Application No. 61-235841, "Suspension of Antigen- or Antibody-Immobilized Magnetic Micro-particles and Method for Measuring the Concentration of Antigen and Antibodies Using Such Suspension" relate to measurements in which an antigen-antibody-magnetic micro-particle combination is produced under the application of a magnetic field, and then application of the magnetic field is stopped and magnetic micro-particles to which unreacted antibodies or antigens are immobilized are dispersed in a sample solution followed by measuring the concentration of the agglutinate suspended in the sample solution. The sensitivity for detection of this method is about 50 micrograms/milliliter. The detection sensitivity of this assay method is about 6 digits lower than that of the method of the present invention described hereinbelow.
Next, earlier proposals on magnetic labeling materials for use in the separation of cells or in the transfer of medicines are summarized below.
(3) Giaever, U.S. Pat. No. 3,970,518, "Magnetic Separation of Biological Particles", discloses a method of separating cells or the like by coating ferromagnetic or ferrimagnetic materials such as ferrite, perovskite, chromite, magnetoplumbite, etc. having a size in the range between the size of colloid particles and 10 micrometers with an antibody.
(4) Davies, et al., U.S. Pat. No. 4,177,253, "Magnetic Particle for Immunoassay", describes composite magnetic particles having a particle size of 1 micrometer to 1 cm and comprising a core material of a low density coated on the surface thereof with a metal magnetic-material such as Ni, etc., and a biologically active substance such as an antigen or antibody.
(5) Molday, U.S. Pat. No. 4,452,773, "Magnetic Iron-Dextran Microspheres", describes dextran-coated micro-particles of magnetite, which is one of ferromagnetic substances having a particle size of preferably 30 to 40 nm.
(6) Czerlinski, U.S. Pat. No. 4,454,234, "Coated Magnetizable Microparticles, Reversible Suspensions Thereof, and Processes Relating Thereto", describes magnetic micro-particles having a particle size in the range between the size of magnetic domain and about 0.1 micrometer and comprising micro-particles of a ferromagnetic material such as ferrite, yttrium-iron-garnet, etc. whose Curie temperature is in the range between 5 degree C. to 65 degree C. and whose surface is coated with a copolymer composition based on acrylamide.
(7) Ikeda, et al., U.S. Pat. No. 4,582,622, "Magnetic Particulate for Immobilization of Biological Protein and Process of Producing the Same", describes particles of a particle size of about 3 micrometers composed mainly of gelatin and containing 0.00001% to 2% ferromagnetic substance composed of ferrite.
(8) Margel, U.S. Pat. No. 4,324,923, "Metal Coated Polyaldehyde Microspheres", describes polyaldehyde microspheres coated with a transient metal and containing ferromagnetic substance such as iron, nickel, cobalt, etc. as a magnetic material.
The magnetic materials described in (4) to (8) above each are ferromagnetic or ferrimagnetic particles having a particle size of at least 30 nm, and are classified under as ferromagnetic materials. Ferromagnetic materials are those having a particle size of usually several tens nm or more, which may vary depending on the kind of the material, and showing residual magnetization after disappearance of an external magnetic field.
However, the proposals on the separation of cells and transfer of medicines are still being researched and most of which have not been put in practice.